Digital affinity linkage assay

ABSTRACT

Methods of detecting a target in a sample are provided. Kits for performing the methods described herein are also provided.

This application claims the benefit of U.S. Provisional Application62/564,029 filed on Sep. 27, 2017 which is hereby incorporated byreference in its entirety.

BACKGROUND

Quantifying the amount of biomolecules in a sample from a subject canprovide useful information for a number of clinical applications. Onemethod for detecting and quantifying biomolecules, such as proteins, isby enzyme-linked immunosorbent assay (ELISA). However, the limit ofdetection and precision of quantification with this assay are notsufficient for many needs. Alternative techniques such as immuno-PCRhave the potential to increase the sensitivity of detection, but inpractice are limited by the problem of high background signal due tonon-specific binding of the antibodies used as detection agents.

SUMMARY

Disclosed herein are methods, compositions, and kits for detecting atarget in a sample.

In an embodiment, a method of detecting a target in a sample comprisescontacting the sample with a first affinity agent linked to a solidsupport, wherein the first affinity agent specifically binds to thetarget, if present, thereby forming a target bound to the first affinityagent; separating the target bound to the first affinity agent fromunbound material in the sample based on the presence or absence of thesolid support, thereby generating a separated sample comprising thetarget bound to the first affinity agent; contacting the separatedsample with a second affinity agent comprising a first label and a thirdaffinity agent comprising a second label, wherein the second and thirdaffinity agents specifically bind to the target, thereby forming atarget-labeled affinity agent complex; separating the target-labeledaffinity agent complex from uncomplexed second and third affinity agentsbased on the presence or absence of the solid support, therebygenerating a separated target-labeled affinity agent complex;partitioning at least the separated target-labeled affinity agentcomplex into a plurality of partitions; and detecting the presence ofthe target in the sample by detecting the presence of the first andsecond labels in at least one same partition.

In some embodiments, the method of detecting a target in a samplecomprises contacting the sample with a first affinity agent linked to asolid support, a second affinity agent comprising a first label, and athird affinity agent comprising a second label, wherein the first,second and third affinity agents specifically bind to the target, ifpresent, thereby forming a target-labeled affinity agent complex;separating the target-labeled affinity agent complex from uncomplexedcomponents in the sample based on the presence or absence of the solidsupport, thereby generating a separated target-labeled affinity agentcomplex; partitioning at least the separated target-labeled affinityagent complex into a plurality of partitions; and detecting the presenceof the target in the sample by detecting the presence of the first andsecond labels in at least one same partition.

In certain embodiments, the method of detecting a target in a samplecomprises contacting the sample with a second affinity agent comprisinga first label and a third affinity agent comprising a second label,wherein the second and third affinity agents specifically bind to thetarget, if present, thereby forming a first complex comprising thetarget, the second affinity agent and third affinity agent; contactingthe first complex with a first affinity agent linked to a solid support,wherein the first affinity agent specifically binds to the target,thereby forming a target-labeled affinity agent complex comprising thetarget, the first affinity agent, the second affinity agent, and thethird affinity agent; separating the target-labeled affinity agentcomplex from uncomplexed components based on the presence or absence ofthe solid support, thereby generating a separated target-labeledaffinity agent complex; partitioning at least the separatedtarget-labeled affinity agent complex into a plurality of partitions;and detecting the presence of the target in the sample by detecting thepresence of the first and second labels in at least one same partition.

In some embodiments, the method of detecting a target in a samplecomprises conjugating a plurality of solid supports to a plurality ofproteins in the sample comprising the target, if present, therebyforming a plurality of solid support conjugates, wherein the targetcomprises at least one of the plurality of proteins; separating theplurality of solid support conjugates from unconjugated material in thesample based on the presence or absence of the plurality of solidsupports, thereby generating a separated plurality of solid supportconjugates; contacting the separated plurality of solid supportconjugates with a first affinity agent comprising a first label and asecond affinity agent comprising a second label, wherein the first andsecond affinity agents specifically bind to the target, thereby forminga target-labeled affinity agent complex; separating the target-labeledaffinity agent complex from uncomplexed first and second affinity agentsbased on the presence or absence of the solid support, therebygenerating a separated target-labeled affinity agent complex;partitioning at least the separated target-labeled affinity agentcomplex into a plurality of partitions; and detecting the presence ofthe target in the sample by detecting the presence of the first andsecond labels in at least one same partition. In some embodiments, theconjugating comprises cross-linking.

In some embodiments, the first affinity agent is cleaved from the solidsupport prior to the partitioning step, thereby releasing thetarget-labeled affinity agent complex from the solid support. In someembodiments, an amino acid tag linking the first affinity agent to thesolid support is cleaved by a sequence-specific protease. In certainembodiments, the protease is TEV, factor Xa, or thrombin. In someembodiments, a photo-cleavable linker between the solid surface and thefirst affinity agent is cleaved by exposing the linker to light.

In some embodiments, the target-labeled affinity agent complex iscross-linked prior to partitioning the separated target-labeled affinityagent complex into a plurality of partitions.

In some embodiments, the first label is a first nucleic acid label andthe second label is a second nucleic acid label. In certain embodiments,the first and second nucleic acid labels are amplified following thepartitioning. In some embodiments, each of the first and second nucleicacid labels are detected using a DNA probe (e.g., a TAQMAN™ probe, aSCORPION™ probe, an ECLIPSE™ probe, a molecular beacon probe, adouble-stranded probe, a dual hybridization probe, or a double-quenchedprobe). In certain embodiments, each of the first and second nucleicacid labels are detected using an intercalating dye (e.g., EvaGreen®dye. In some embodiments, the first and second nucleic acid labels aredetected using different signal levels of the same DNA probe orintercalating dye. In some embodiments, the first label is a firstfluorophore and the second label is a second fluorophore. In certainembodiments, the first label is a first enzyme, the second label is asecond enzyme, and the detecting comprises detecting products generatedby the first and second enzymes. In some embodiments, the first labelgenerates a first signal and the second label generates a second signaland the first signal and the second signal are distinguishable.

In some embodiments, the first label and the second label are eachlinked to streptavidin, the second and third affinity agents arebiotinylated, and the second and third affinity agents are labeled withthe streptavidin-linked first and second labels, respectively, prior toa first step of the method by allowing the first and second labeledstreptavidins to bind to the respective biotinylated second and thirdaffinity agents (i.e., by streptavidin-biotin interaction).

In some embodiments, the first affinity agent is biotinylated and thesolid support is linked to streptavidin, and prior to a first step ofthe method the streptavidin-linked solid support is linked to the firstaffinity agent by allowing the streptavidin-linked solid support to bindto the biotinylated first affinity agent (i.e., by streptavidin-biotininteraction).

In certain embodiments, the solid support or the plurality of solidsupports is/are a magnetic bead(s), a non-magnetic bead(s), or asurface(s) of a reaction vessel(s). In some embodiments, thenon-magnetic beads are polystyrene beads or silica-based beads. In someembodiments, the solid support or the plurality of solid supports aremagnetic beads and the target-labeled affinity agent complex isseparated from the uncomplexed components in the sample using a magnetthat attracts the magnetic beads linked to the first affinity agent inthe target-labeled affinity agent complex. In certain embodiments, thesolid support or the plurality of solid supports are non-magnetic beadsand the target-labeled affinity agent complex is separated from theuncomplexed components in the sample by centrifugation. In someembodiments, the solid support is a surface of a reaction, and thetarget-labeled affinity agent complex is separated from the uncomplexedcomponents in the sample by aspiration.

In certain embodiments, the target comprises a protein, a proteinaggregate, or a protein oligomer. In some embodiments, the target is acomplex of two or more interacting proteins and the second and thirdaffinity agents each bind to one of the interacting proteins in thecomplex. In some embodiments, the target has a repeating identicalepitope and the first and second affinity agents or the first and thirdaffinity agents recognize the same epitope. In some embodiments, two ormore of the first, second, and third affinity agents recognize adifferent epitope on the target. In some embodiments, each of the first,second, and third affinity agents recognizes a different epitope on thetarget.

In some embodiments, the first, second, and third affinity agents areeach selected from the group consisting of an antibody, an antibodyfragment, and a nucleotide aptamer. In some embodiments, the antibody isa monoclonal antibody and/or a polyclonal antibody.

In some embodiments (e.g., a multiplexing embodiment), the target is aplurality of different targets, the first affinity agent is a pluralityof different first affinity agents, the second affinity agent is aplurality of different second affinity agents comprising a plurality offirst labels, and the third affinity agent is a plurality of differentthird affinity agents comprising a plurality of second labels andwherein a set of the first, second and third affinity agentsspecifically bind to the same one of the plurality of different targets.

In certain embodiments, the method further comprises determining thenumber of partitions comprising the first label and the second label,thereby quantifying the target. In some embodiments, the partitions aredroplets.

In an embodiment, a kit for detecting a target in a sample comprises afirst affinity agent linked to a solid support; a second affinity agentcomprising a first label; and a third affinity agent comprising a secondlabel, wherein each of the first, second, and third affinity agentsspecifically binds to the target. In some embodiments, the second andthird affinity agents specifically bind to different epitopes on thetarget than the first affinity agent. In some embodiments, a kit fordetecting a target in a sample comprises a chemically modified solidsupport; a first label; and a second label, wherein each of first andsecond labels is linked to streptavidin.

In some embodiments in which the first label is a first oligonucleotideand the second label is a second oligonucleotide, the kit furthercomprises a DNA polymerase, PCR primers, PCR probes, dNTPs, a buffer,and/or a PCR master mix. In some embodiments in which the first label isa first enzyme and the second label is a second enzyme, the kit furthercomprises at least one of a first and second substrate for therespective first and second enzymes, and/or a buffer. In certainembodiments, the kit further comprises instructions for performing amethod of detecting a target in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing a method of detecting a target in asample according to an embodiment of the invention.

FIG. 2 describes the workflow of Example 1. Capture beads coated withthe first affinity agent were reacted with the sample comprising thetarget antigen. A wash was performed to remove unbound components, thenthe target-bound beads were reacted with two different DNA-labeledaffinity agents (“probes”). Some of the probes bound to the target-boundbeads and the unbound probes were washed away. The complexes comprisingthe bead, target antigen, and labeled probes were then resuspended in asolution comprising an amplification mix and partitioned into droplets.ddPCR was performed and droplet fluorescence was measured. Linkage wasdetermined according to the number of double positive droplets.

FIG. 3 shows dose response curves for an experiment as described inExample 1 (quantitation of a target protein using monoclonalantibodies). The results from two individual immuno-PCR assays (triangleand square markers with dashed curves) were compared to an affinitylinkage assay with the same two DNA-labeled monoclonal antibodies(circle markers with solid curve). The vertical lines mark thecalculated Limit of Detection (LOD) values.

FIGS. 4A-4C show dose response curves for experiments as described inExample 2 (quantitation of different target proteins using polyclonalantibodies). For each target protein, the results from two individualimmuno-PCR assays (triangle and square markers with dashed curves) werecompared to an affinity linkage assay with the same two DNA-labeledpolyclonal antibodies (circle markers with solid curve). The verticallines mark the calculated LOD values for the individual Immuno-PCRassays vs. the digital affinity linkage assays.

FIG. 5 illustrates a proposed workflow for multiplex proteinquantitation of different targets with a digital affinity linkage assay.

FIGS. 6A-6C show intra-assay comparison of dose response curves forexperiments as described in Examples 2 and 3, quantitation of differenttarget proteins using either singleplex or multiplex linkage assays. Foreach target protein, the results from a singleplex assay (square markerswith dashed curves) were compared to multiplex assay (circle markerswith solid curve). The vertical lines mark the calculated LOD values forthe singleplex assays vs. the multiplex assays.

DETAILED DESCRIPTION I. Introduction

Described herein are methods of detecting a target in a sample. Digitalaffinity linkage assay methods have been discovered in which theconcentration of a target is determined on a solid support by separatingthe sample into small partitions (e.g., droplets) and performing digitalPCR analysis of two different DNA-labeled affinity agents (e.g.DNA-labeled antibodies). A linkage signal is calculated from thefraction of partitions in which the two different DNA labels areamplified and co-localized in the same partition.

As described herein, it has been surprisingly discovered that by using asolid support linked to an affinity agent in order to capture a targetand wash away components other than the target-affinity agent complex,in combination with the use of two different labeled detection affinityagents and the subsequent linkage signal from co-localization of the twodifferent labeled affinity agents for detection of a target, thebackground noise from non-specific binding of the affinity agents can besignificantly reduced. Accordingly, the digital affinity linkage assaymethods described herein have reduced background noise from non-specificbinding of DNA-labeled affinity agents and have an unexpectedly highdetection sensitivity or lower limit of detection. The methods describedherein thus provide advantages such as the ability to detectsignificantly lower amounts of target in a sample as compared to assaysin which targets are individually detected, and reducing or eliminatingthe need for diluting the concentration of affinity agents in a reactionmixture, thus avoiding the problem of diluting the affinity agents belowthe detection concentration.

II. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4th ed. 2007); Green et al., MOLECULAR CLONING, ALABORATORY MANUAL (FOURTH EDITION), Cold Spring Harbor Lab Press (ColdSpring Harbor, N.Y. 2012).

The term “a” or “an” is intended to mean “one or more.” The term“comprise,” and variations thereof such as “comprises” and “comprising,”when preceding the recitation of a step or an element, are intended tomean that the addition of further steps or elements is optional and notexcluded. Any methods, devices and materials similar or equivalent tothose described herein can be used in the practice of this invention.The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

The term “target” refers to any agent whose presence and/or amount is tobe determined. In some embodiments, the target can be a mixture ofseveral targets. In some embodiments the presence or concentrationprofile of the different targets is to be determined.

The target can be any biological and/or chemical agent (e.g., amolecule, macromolecule, complex, or conjugate). In some embodiments thetarget is an organic or inorganic molecule. In some embodiments thetarget is a biological agent.

In one embodiment the target is a protein, a protein aggregate, aprotein oligomer, a polypeptide or a peptide. In some embodiments, theprotein aggregate comprises more than one protein (e.g., more than onetarget). In some embodiments, the protein has more than one identical ornon-identical subunit which may or may not be covalently bound to eachother. The targets can be hormones, antibodies, amino acids (e.g.,glutamic acid, aspartic acid) or any derivatives and/or combinationthereof. In some embodiments the target is a toxin or a drug. In someembodiments, the amount of the target in the sample is in micrograms. Insome embodiments, the amount of the target in the sample is below 1microgram. In some embodiments, amount of the target in the sample is innanograms. In some embodiments the amount of target in the sample isbetween 100 ng to 1 ng. In some embodiments, the amount of target in thesample is between 1000 pg to 1 pg. In certain embodiments, the amount oftarget in the sample is between 1 pg to 1 fg. In certain embodiments,the amount of target in the sample is between 1 fg to 1 ag.

The term “affinity agent” refers to a molecule that specifically bindsto a target. Exemplary affinity agents include, but are not limited to,an antibody, an antibody fragment, a non-antibody protein scaffold, anantibody mimetic, or an aptamer.

The term “antibody” refers to a polypeptide of the immunoglobulin familyor a polypeptide comprising fragments of an immunoglobulin that iscapable of noncovalently, reversibly, and in a specific manner bindingto a corresponding target (or antigen). The term includes, but is notlimited to, polyclonal or monoclonal antibodies of the isotype classesIgA, IgD, IgE, IgG, and IgM, derived from human or other mammaliancells, including natural or genetically modified forms such ashumanized, human, single-chain, chimeric, synthetic, recombinant,hybrid, mutated, grafted, and in vitro generated antibodies. The termencompasses conjugates, including but not limited to fusion proteinscontaining an immunoglobulin moiety (e.g., chimeric or bispecificantibodies or single chain Fv's (scFv's)), and fragments, such as Fab,F(ab′)2, Fv, scFv, Fd, dAb and other compositions.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively. The variable region contains the antigen-bindingregion of the antibody (or its functional equivalent) and is mostcritical in specificity and affinity of binding. See Paul, FundamentalImmunology (2003).

Antibodies can exist as intact immunoglobulins or as any of a number ofwell-characterized fragments that include specific antigen-bindingactivity. Such fragments can be produced by digestion with variouspeptidases. Pepsin digests an antibody below the disulfide bonds in thehinge region to produce F(ab)′₂, a dimer of Fab which itself is a lightchain joined to V_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ can bereduced under mild conditions to break the disulfide bond in the hingeregion, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. TheFab′ monomer is essentially Fab with part of the hinge region. Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments can besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., scFv) or those identified using phage displaylibraries (see, e.g., McCafferty et al. (1990) Nature 348:552-554).Methods for the preparation of antibodies are known in the art; see,e.g., Kohler & Milstein (1975) Nature 256:495-497; Kozbor et al. (1983)Immunology Today 4:72; Cole et al., Monoclonal Antibodies and CancerTherapy, pp. 77-96. Alan R. Liss, Inc. 1985).

As used herein, the term “F_(V)” refers to a monovalent or bi-valentvariable region fragment, and can encompass only the variable regions(e.g., V_(L) and/or V_(H)), as well as longer fragments, e.g., an Fab,Fab′ or F(ab′)2, which also includes C_(L) and/or C_(H)1. Unlessotherwise specified, the term “Fc” refers to a heavy chain monomer ordimer comprising C_(H)1 and C_(H)2 regions.

The term “binds,” as used with respect to an affinity agent binding toan antigen, typically indicates that the affinity agent (e.g., anantibody) binds a majority of the antigen in a pure population, assumingan appropriate molar ratio of affinity agent to antigen. For example, anaffinity agent that binds a given antigen typically binds to at least ⅔of the antigen molecules in a solution (e.g., 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). One of skill willrecognize that some variability will arise depending on the methodand/or threshold of determining binding.

The term “specifically binds to,” as used with reference to an affinityagent, refers to an affinity agent (e.g., an antibody) that binds to anantigen with at least 2-fold greater affinity than to non-antigenmolecules, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold. 10⁶-fold, 10⁷-fold, 10⁶-fold, 10⁹-fold, 10¹⁰-fold,10¹¹-fold, 10¹²-fold, 10¹³-fold, 10¹⁴-fold, or 10¹⁵-fold greateraffinity. For example, an affinity agent that specifically binds aparticular antigen will typically bind the antigen with at least a2-fold greater affinity than to a non-antigen molecule.

As used herein, “nucleic acid” means a compound comprising a chain ofnucleotide monomers. A nucleic acid can be single-stranded ordouble-stranded (i.e., base-paired with another nucleic acid), amongothers. The chain of a nucleic acid can be composed of any suitablenumber of monomers, such as at least about ten or one hundred, amongothers. Generally, the length of a nucleic acid chain corresponds to itssource, with synthetic nucleic acids (e.g., nucleic acid reagents suchas primers and probes) typically being shorter and biologically producednucleic acids (e.g., nucleic acid analytes) typically being longer.

A nucleic acid can have a natural or artificial structure, or acombination thereof. Nucleic acids with a natural structure, namely,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), have a backboneof alternating pentose sugar groups and phosphate groups. Each pentosegroup is linked to a nucleobase (e.g., a purine (such as adenine (A) orguanine (T)) or a pyrimidine (such as cytosine (C), thymine (T), oruracil (U))). Nucleic acids with an artificial structure are analogs ofnatural nucleic acids and can, for example, be created by changes to thepentose and/or phosphate groups of the natural backbone. Exemplaryartificial nucleic acids include glycol nucleic acids (GNA), peptidenucleic acids (PNA), locked nucleic acid (LNA), threose nucleic acids(TNA), and the like. Similarly, nucleic acids having an artificialstructure are analogs of natural nucleic acids and can, for example, becreated by changes to the nucleobase. Exemplary artificial ornon-naturally occurring nucleobases include, but are not limited tohalogenated nucleobases (5-FU), hypoxanthine, xanthine, 7-methylguanine,inosine, xanthosine, 7-methyguanosine, 5,6-dihydrouracil,5-methycytosine, 5-hydroxymethylcytosine, dihydrouridine, and5-methylcytidine.

The sequence of a nucleic acid is defined by the order in whichnucleobases are arranged along the backbone (typically read from the 5′to 3′ end). This sequence generally determines the ability of thenucleic acid to bind specifically to a partner chain (or to form anintramolecular duplex) by hydrogen bonding. In particular, adenine pairswith thymine uracil) and guanine pairs with cytosine. A nucleic acidthat can bind to another nucleic acid in an antiparallel fashion byforming a consecutive string of adenine-thymine and guanine-cytosinebase pairs with the other nucleic acid is termed “complementary.”

The terms “label”, “detectable label”, or “labeling agent” refer to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include fluorescent dyes (fluorophores), fluorescent quenchers,luminescent agents, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, ³²P and other isotopes, haptens,proteins, nucleic acids, or other substances which can be madedetectable, e.g, by incorporating a label into an oligonucleotide,peptide, or antibody specifically reactive with a target molecule. Theterm includes combinations of single labeling agents, e.g., acombination of fluorophores that provides a unique detectable signature,e.g., at a particular wavelength or combination of wavelengths. Adetectable label can also include a combination of a reporter and aquencher.

A molecule that is “linked” to a label (e.g., as for a labeled nucleicacid as described herein) is one that is bound, either covalently,through a linker or a chemical bond, or noncovalently, through ionic,van der Waals, electrostatic, or hydrogen bonds to a label such that thepresence of the molecule can be detected by detecting the presence ofthe label bound to the molecule.

The term “reporter” refers to a substance or a portion thereof which iscapable of exhibiting a detectable signal, which signal can besuppressed by a quencher. The detectable signal of the reporter is,e.g., fluorescence in the detectable range; thus, a reporter can also bea label.

The term “quencher” refers to a substance which is capable ofsuppressing, reducing, inhibiting, etc., the detectable signal producedby the reporter.

As used herein, the term “quenching” refers to a process whereby, when areporter and a quencher are in close proximity, and the reporter isexcited by an energy source, a substantial portion of the energy of theexcited state non-radiatively transfers to the quencher where it eitherdissipates nonradiatively or is emitted at a different emissionwavelength than that of the reporter (e.g., by fluorescence resonanceenergy transfer or FRET).

The reporter can be selected from fluorescent organic dyes modified witha suitable linking group for attachment to the oligonucleotide, such asto the 3′ or 5 terminus. The quencher can also be selected from organicdyes, which may or may not be fluorescent, depending on the embodimentof the invention. Generally, whether the quencher is fluorescent orsimply releases the transferred energy from the reporter bynon-radiative decay, the absorption band of the quencher should at leastsubstantially overlap the fluorescent emission band of the reporter tooptimize the quenching.

Non-fluorescent quenchers or dark quenchers typically function byabsorbing energy from excited reporters, but do not release the energyradiatively.

Selection of appropriate reporter-quencher pairs for particular probescan be undertaken in accordance with known techniques. Fluorescent anddark quenchers and their relevant optical properties from whichexemplary reporter-quencher pairs can be selected are listed anddescribed, for example, in R. W. Sabnis, HANDBOOK OF FLUORESCENT DYESAND PROBES, John Wiley and Sons, New Jersey, 2015, the content of whichis incorporated herein by reference.

Reporter-quencher pairs can be selected from xanthene dyes includingfluoresceins and rhodantine dyes. Many suitable forms of these compoundsare available commercially with substituents on the phenyl groups, whichcan be used as the site for bonding or as the bonding functionality forattachment to an oligonucleotide. Another group of fluorescent compoundsfor use as reporters are the naphthylamines, having an amino group inthe alpha or beta position. Included among such naphthylamino compoundsare 1-dimethylaminonaphthy 1-5 sulfonate, 1-anilino-8-naphthalenesulfonate and 2-p-touidiny 1-6-naphthalene sulfonate. Other dyes include3-phenyl-7-isocyanatocoumarin; acridines such as9-isothiocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide;benzoxadiazoles; stilbenes; pyrenes and the like.

Suitable examples of quenchers can be selected from6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo) benzoicacid (DABYL), tetramethylrhodamine (TAMRA), BHQ-OTM, BHQ-1 TM, BHQ-2TM,and BHQ-3TM, each of which are available from Biosearch Technologies,Inc. of Novato, Calif., Qy7TM QSY-9TM, QSY-21 TM and QSY-35TM, each ofwhich are available from Molecular Probes, Inc, and ZEN™ and TAO™Double-Quenched Probes from Integrated DNA Technologies.

Suitable examples of reporters can be selected from dyes such as SYBRgreen, 5-carboxyfluorescein (5-FAM™ available from Applied Biosystems ofFoster City, Calif.), 6-carboxyfluorescein (6-FAM),tetrachloro-6-carboxyfluorescein (TET),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein,hexachloro-6-carboxyfluorescein (HEX),6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TET™ available fromApplied Biosystems), carboxy-X-rhodamine (ROX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOETM availablefrom Applied Biosystems), VICTM dye products available from MolecularProbes, Inc., NEDTM dye products available from Applied Biosystems, CalFluor dye products (such as, e.g., Cal Fluor Gold 540, Orange 560, Red590, Red 610, Red 635) available from Biosearch Technologies, Quasar dyeproducts (such as, e.g., Quasar 570, 670, 705) available from BiosearchTechnologies, and the like.

The term “partitioning” or “partitioned” refers to separating an aqueoussolution having one or more of a sample and reactant into a plurality ofportions, or “partitions.” Partitions can be solid or fluid. In someembodiments, a partition is a solid partition, e.g., a microchannel, amicrotube, or a microwell. In some embodiments, a partition is a fluidpartition, e.g., a droplet. In some embodiments, a fluid partition (e.g,a droplet) is a mixture of immiscible fluids (e.g., water and oil). Insome embodiments, a fluid partition (e.g., a droplet) is an aqueousdroplet that is surrounded by an immiscible carrier fluid oil).

III. Detection Methods

A method 100 of detecting a target in a sample will now be described.Some of the steps can be performed in any suitable order, in anysuitable combination, and can be combined with or modified by any othersuitable aspects of the disclosure provided herein.

In exemplary step 110, the sample is contacted with a first affinityagent (e.g., an antibody or antibody fragment) linked to a solidsupport. The first affinity agent specifically binds to a first epitopeon the target, if present, thereby forming a target bound to the firstaffinity agent. In some embodiments, the sample is incubated with thefirst affinity agent to allow time for the first affinity agent tocapture or bind to the target. In some embodiments, the sample isincubated with the first affinity agent for one hour or more. In someembodiments, prior to contacting the sample with the first affinityagent linked to the solid support, the solid support is treated with ablocking agent to prevent non-specific, binding of material to thesupport. Exemplary blocking agents include, but are not limited to,proteins (e.g., non-fat milk or bovine serum albumin) and detergents(e.g., Tween 20 or Triton X-100).

In some embodiments, the sample is a biological sample. Biologicalsamples can be obtained from any biological organism, e.g., an annual,plant, fungus, bacterial, or any other organism. In some embodiments,the biological sample is from an animal, e.g., a mammal (e.g., a humanor a non-human primate, a cow, horse, pig, sheep, cat, dog, mouse, orrat), a bird (e.g., chicken), or a fish. A biological sample can be anytissue or bodily fluid obtained from the biological organism, e.g.,blood, a blood fraction, or a blood product (e.g., serum, plasma,platelets, red blood cells, and the like), sputum or saliva, tissue(e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye,skeletal muscle, cartilage, or bone tissue); cultured cells, e.g.,primary cultures, explants, transformed cells, stem cells, bacterialcells, stool, or urine.

In some embodiments, the sample can be prepared to improve the efficientdetection of the target(s). For example, in some embodiments the samplecan be fragmented, fractionated, homogenized, or sonicated. In someembodiments, a target of interest, or a sub-fraction comprising thetarget of interest, can be extracted or isolated from a sample (e.g., abiological sample). In some embodiments, the sample is enriched for thepresence of the one or more targets. In some embodiments, the target isenriched in the sample by an affinity method, e.g., immunoaffinityenrichment. In some embodiments, the target is enriched in the sampleusing size selection (e.g., removing very small fragments or moleculesor very long fragments or molecules).

Exemplary solid supports include, but are not limited to, particles(e.g., magnetic beads, polymeric beads, or silica-based beads) or asolid surface the surface of a reaction vessel such as a tube or a wellin a plate). In some embodiments, the solid support is not chemicallymodified prior to the attachment of the first affinity agent antibody(e.g., the antibody is attached to the substrate by non-covalentadsorption, based on hydrophobic and other interactions). In certainembodiments, the solid support is chemically modified prior to theattachment of the first affinity agent. Exemplary chemically modifiedsolid supports can have carboxyl or amine groups attached and thesegroups can be used to covalently bind the first affinity agent. In someembodiments, the first affinity agent is attached to the solid supportvia carbodiimide mediated chemistry to form an amide bond. In someembodiments, the attachment of the first affinity agent to the solidsupport is enhanced by a chemical or a photochemical reaction. In someembodiments, the first affinity agent is biotinylated and is attached tothe solid support via an avidin-biotin or streptavidin-biotininteraction. In certain embodiments, the first affinity agent ispermanently attached to the solid support by a chemical or aphotochemical reaction. In some embodiments, the solid support isdeactivated after attaching the first affinity agent to the solidsupport to prevent binding of other agents. For example, active carboxylgroups on the solid support can be deactivated with ethanolamine.

In exemplary step 120, the target bound to the first affinity agent isseparated (e.g., the solid support is washed with a wash solution) fromunbound material in the sample based on the presence or absence of thesolid support, thereby generating a separated sample comprising thetarget bound to the first affinity agent. In some embodiments, a buffercomprising a detergent such as Tween 20 or Triton X-100 is used toremove or separate unbound material from the solid support. In someembodiments in which the solid support is a magnetic bead, the targetbound to the first affinity agent is separated from the uncomplexedcomponents in the sample using a magnet that attracts the magnetic beadslinked to the first affinity agent. In some embodiments in which thesolid support is a non-magnetic bead (e.g., polystyrene or silica-basedbeads), the target bound to the first affinity agent is separated fromthe uncomplexed components in the sample by centrifugation. In someembodiments in which the solid support is a surface of a reaction vessel(e.g., a tube or a well), the target bound to the first affinity agentis separated from the uncomplexed components in the sample byaspiration.

In exemplary step 130, the separated sample is contacted with a secondaffinity agent comprising a first label and a third affinity agentcomprising a second label, wherein the second and third affinity agentsspecifically bind to the target, thereby forming a target-labeledaffinity agent complex. In some embodiments, the second and thirdaffinity agents specifically bind to different epitopes on the targetthan the first affinity agent. In some embodiments, the target (e.g., adimeric protein, an aggregate-forming protein, or an oligomeric protein)has a repeating identical epitope such that the first and secondaffinity agents or the first and third affinity agents recognize thesame epitope. In some embodiments, the target is a complex of two ormore interacting proteins and the second and third affinity agents eachbind to one of the interacting proteins in the complex. In certainembodiments, the target is a complex of two or more interacting partners(e.g., two proteins, a protein and a non-protein molecule(s), ornon-protein molecules) and the method further comprises calculating abinding affinity (KD) of the interacting partners.

In certain embodiments (e.g., a multiplexing embodiment), the samplecomprises a plurality of different targets, and for each of theplurality of different targets in the sample, a set of first, second,and third affinity agents is provided wherein each of the first, second,and third affinity agents specifically binds to the target. Thus, thefirst affinity agent is a plurality of different first affinity agents(i.e., each of the different first affinity agents binding to adifferent target in the sample), the second affinity agent is aplurality of different second affinity agents comprising a plurality offirst labels (i.e., each of the different second affinity agents bindingto a different target in the sample), and the third affinity agent is aplurality of different third affinity agents comprising a plurality ofsecond labels (i.e., each of the different third affinity agents bindingto a different target in the sample). A set of the first, second andthird affinity agents specifically bind to the same one of the pluralityof different targets.

In some embodiments, the first, second, and third affinity agents areantibodies, antibody fragments, or nucleotide aptamers. In certainembodiments, the antibodies are monoclonal antibodies or polyclonalantibodies.

In exemplary step 140, the target-labeled affinity agent complex isseparated (e.g., the solid support is again washed) from uncomplexedsecond and third affinity agents based on the presence or absence of thesolid support, thereby generating a separated target-labeled affinityagent complex.

In some embodiments, the first affinity agent is cleaved from the solidsupport prior to the next (partitioning) step, thereby releasing thetarget-labeled affinity agent complex from the solid support. In someembodiments, an amino acid tag linking the target-labeled affinity agentcomplex to the solid support is cleaved by a sequence-specific protease(e.g., TEV, factor Xa, or thrombin). In certain embodiments, aphoto-cleavable linker between the solid surface and the first affinityagent is cleaved by exposing the linker to light. In some embodiments,the target-labeled affinity agent complex is cross-linked prior topartitioning the separated target-labeled affinity agent complex into aplurality of partitions.

In exemplary step 150, a plurality of partitions are formed from theseparated target-labeled affinity agent complex such that a subset ofthe partitions contains the target-labeled affinity agent complex. Thepartitions can include any of a number of types of partitions, includingsolid partitions (e.g., wells or tubes) and fluid partitions (e.g.,aqueous phase or droplet within an oil phase). In some embodiments, thepartitions are droplets. In some embodiments, the partitions aremicrochannels or microwells. Methods and compositions for partitioning asolution are described, for example, in published patent applications WO2012/135259, WO 2014/117088, WO 2010/036352, and U.S. Pat. No.9,156,010, the entire content of each of which is incorporated byreference herein.

In some embodiments, a droplet comprises an emulsion composition, i.e.,a mixture of immiscible fluids (e.g., water and oil). In someembodiments, a droplet is an aqueous droplet that is surrounded by animmiscible carrier fluid (e.g., oil). In some embodiments, a droplet isan oil droplet that is surrounded by an immiscible carrier fluid (e.g.,an aqueous solution). In some embodiments, the droplets described hereinare relatively stable and have minimal coalescence between two or moredroplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%,0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10% of droplets generated coalesce with other droplets.

In an embodiment, the droplet is formed by flowing an oil phase throughan aqueous phase. The oil for the oil phase can be synthetic ornaturally occurring. In some embodiments, the oil comprises carbonand/or silicon. In some embodiments, the oil comprises hydrocarbonand/or fluorocarbon. Exemplary oils include, but are not limited to,silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or acombination thereof.

The oil phase can comprise a fluorinated base oil which can additionallybe stabilized by combination with a fluorinated surfactant such as aperfluorinated polyether. In some embodiments, the base oil comprisesone or more of a HFE 7500, FC-40, FC-43, FC-70, or another commonfluorinated oil. In some embodiments, the oil phase comprises an anionicfluorosurfactant. In some embodiments, the anionic fluorosurfactant isAmmonium Krytox (Krytox-AS), the ammonium salt of Krytox FSH, or amorpholino derivative of Krytox FSH. Krytox-AS can be present at aconcentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments, theconcentration of Krytox-AS is about 1.8%. In some embodiments, theconcentration of Krytox-AS is about 1.62%. Morpholino derivative ofKrytox FSH may be present at a concentration of about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.8%. In some embodiments, the concentration of morpholinoderivative of Krytox FSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0% 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

In some embodiments, the droplet is formed by flowing an oil phasethrough an aqueous solution phase having a DNA template and one or morecomponents (e.g., reagents) that are used to determine the presence orabsence of the target. In some embodiments, the one or more componentsused to determine the presence or absence of the target in the aqueousdroplet are soluble and/or miscible in water including, but not limitedto, one or more salts, buffering agents, reagents (e.g., a releasingagent such as a restriction endonuclease or a protease, PCR components),surfactants, and/or whatever additional components are necessary for adesired reaction(s) that is intended to occur within a formed droplet.All such additional components can be selected to be compatible with thedesired reaction or intended assay.

In some embodiments, assay components (e.g., a DNA polymerase, dNTPs,and/or a PCR master mix, enzyme substrates) can be injected into thepartition. The assay components can be injected into the partition inany order or simultaneously.

Methods of injecting fluids into partitions are described in, forexample, WO 2012/135259 and US 2012/0132288, each of which isincorporated by reference in its entirety.

In some embodiments, at least 500 partitions (e.g., droplets), at least1000 partitions, at least 2000 partitions, at least 3000 partitions, atleast 4000 partitions, at least 5000 partitions, at least 6000partitions, at least 7000 partitions, at least 8000 partitions, at least10,000 partitions, at least 15,000 partitions, at least 20,000partitions, at least 30,000 partitions, at least 40,000 partitions, atleast 50,000 partitions, at least 60,000 partitions, at least 70,000partitions, at least 80,000 partitions, at least 90,000 partitions, atleast 100,000 partitions, at least 200,000 partitions, at least 300,000partitions, at least 400,000 partitions, at least 500,000 partitions, atleast 600,000 partitions, at least 700,000partitions, at least 800,000partitions, at least 900,000 partitions, at least 1,000,000 partitions,at least 2,000,000 partitions, at least 3,000,000 partitions, at least4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000partitions, at least 20,000,000 partitions, at least 30,000,000partitions, at least 40,000,000 partitions, at least 50,000,000partitions, at least 60,000,000 partitions, at least 70,000,000partitions, at least 80,000,000 partitions, at least 90,000,000partitions, at least 100,000,000 partitions, at least 150,000,000partitions, or at least 200,000,000 partitions are formed.

In some embodiments, the droplets that are generated are substantiallyuniform in shape and/or size. For example, in some embodiments, thedroplets are substantially uniform in average diameter. The term“substantially” or “about” refers to the recited number and any valuewithin 10% of the recited number. In some embodiments, the droplets thatare generated have an average diameter of about 0.001 microns, about0.005 microns, about 0.01 microns, about 0.05 microns, about 0.1microns, about 0.5 microns. about 1 microns, about 5 microns, about 10microns, about 20 microns, about 30 microns, about 40 microns, about 50microns, about 60 microns, about 70 microns, about 80 microns, about 90microns, about 100 microns, about 150 microns, about 200 microns, about300 microns, about 400 microns, about 500 microns, about 600 microns,about 700 microns, about 800 microns, about 900 microns, or about 1000microns. In some embodiments, the droplets that are generated have anaverage diameter of less than about 1000 microns, less than about 900microns, less than about 800 microns, less than about 700 microns, lessthan about 600 microns, less than about 500 microns, less than about 400microns, less than about 300 microns, less than about 200 microns, lessthan about 100 microns, less than about 50 microns, or less than about25 microns. In some embodiments, the droplets that are generated arenon-uniform in shape and/or size.

In some embodiments, the droplets that are generated are substantiallyuniform in volume. For example, in some embodiments, the droplets thatare generated have an average volume of about 0.001 nL, about 0.005 nL,about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL,about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL,about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL,about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, orabout 50 nL.

In some embodiments, the partitions (e.g., droplets) are stable and arecapable of long-term storage. In some embodiments, the partitions arestored at about −70°, −20°, 0°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°,20°, 25°, 30°, 35°, or 40° C. for an extended period of time (e.g., forat least 30 days, at least 60 days, at least 90 days, or longer).

Partitions as described herein can contain one or more surfactants toreduce coalescence of droplets during transport. As used herein, a“surfactant” is a surface-active substance capable of reducing thesurface tension of a liquid in which it is present. A surfactant, whichalso or alternatively is described as a detergent and/or a wettingagent, can incorporate both a hydrophilic portion and a hydrophobicportion, which can collectively confer a dual hydrophilic-hydrophobiccharacter on the surfactant. A surfactant can, in some cases, becharacterized according to its hydrophilicity relative to itshydrophobicity. In some embodiments, the aqueous phase incorporates atleast one hydrophilic surfactant. The aqueous phase can include at leastone nonionic surfactant and/or ionic surfactant. In certain embodiments,the aqueous phase includes a surfactant that is a block copolymer ofpolypropylene oxide and polyethylene oxide. In some embodiments, thesurfactant is a block copolymer of polypropylene oxide and polyethyleneoxide sold under the trade names PLURONIC and TETRONIC (BASF). In someembodiments, the surfactant is a nonionic block copolymer ofpolypropylene oxide and polyethylene oxide sold under the trade namePLURONIC F-68. In some embodiments, the surfactant of the aqueous phaseis a water-soluble and/or hydrophilic fluorosurfactant. Exemplaryfluorosurfactants for the aqueous phase are sold under the trade nameZONYL (DuPont), such as ZONYL FSN fluorosurfactants. In some cases, thesurfactant can include polysorbate 20 (sold under the trade nameTWEEN-20 by ICI Americas, Inc.). The concentration of a particularsurfactant or total surfactant present in the aqueous phase can beselected to stabilize emulsion droplets prior to heating. In someembodiments, the concentration of surfactant for the aqueous phase is0.01 to 10%, 0.05 to 5%, 0.1 to 1%, or 0.5% by weight.

In exemplary step 160, the presence of the target in the sample isdetected by detecting the presence of the first and second labels in atleast one same partition (i.e., the first and second labels areco-located in the same partition).

In certain embodiments, a method of detecting a target in a samplecomprises contacting the sample with a first affinity agent linked to asolid support, a second affinity agent comprising a first label, and athird affinity agent comprising a second label. The first, second andthird affinity agents specifically bind to the target, if present,thereby forming a target-labeled affinity agent complex. In someembodiments, the first, second, and third affinity agents specificallybind to different epitopes on the target. The next step comprisesseparating the target-labeled affinity agent complex from uncomplexedcomponents in the sample based on the presence or absence of the solidsupport, thereby generating a separated target-labeled affinity agentcomplex. Next, the separated target-labeled affinity agent complex ispartitioned into a plurality of partitions. The presence of the targetis detected in the sample by detecting the presence of the first andsecond labels in at least one same partition.

In some embodiments, a method of detecting a target in a samplecomprises contacting the sample with a second affinity agent comprisinga first label and a third affinity agent comprising a second label. Thesecond and third affinity agents specifically bind to the target, ifpresent, thereby forming a first complex comprising the target, thesecond affinity agent, and third affinity agent. In some embodiments,the second and third affinity agents specifically bind to differentepitopes on the target. The next step of the method comprises contactingthe first complex with a first affinity agent linked to a solid support.The first affinity agent specifically binds to the target, therebyforming a target-labeled affinity agent complex comprising the target,the first affinity agent, the second affinity agent, and the thirdaffinity agent. In some embodiments, the first affinity agentspecifically binds to a different epitope on the target than the secondand third affinity agents. Next, the target-labeled affinity agentcomplex is separated from uncomplexed components based on the presenceor absence of the solid support, thereby generating a separatedtarget-labeled affinity agent complex. At least the separatedtarget-labeled affinity agent complex is then partitioned into aplurality of partitions. The presence of the target in the sample isdetected by detecting the presence of the first and second labels in atleast one same partition.

In certain embodiments, a method of detecting a target in a samplecomprises conjugating a plurality of solid supports to a plurality ofproteins in the sample comprising the target, if present, therebyforming a plurality of solid support conjugates. In some embodiments,the conjugating step comprises cross-linking. Cross-linking methods andreagents are known in the art, and include but are not limited to theuse of carbodiimides, pryidyl disulfides, carbonyls, hydrazides,maleimides, haloacetyls, and diazirines. The next step comprisesseparating the plurality of solid support conjugates from unconjugatedmaterial in the sample based on the presence or absence of the pluralityof solid supports, thereby generating a separated plurality of solidsupport conjugates. Next, the separated plurality of solid supportconjugates are contacted with a first affinity agent comprising a firstlabel and a second affinity agent comprising a second label. The firstand second affinity agents specifically bind to different epitopes onthe target, thereby forming a target-labeled affinity agent complex. Inthe next step, the target-labeled affinity agent complex is separatedfrom uncomplexed first and second affinity agents based on the presenceor absence of the solid support, thereby generating a separatedtarget-labeled affinity agent complex. Next, at least the separatedtarget-labeled affinity agent complex is partitioned into a plurality ofpartitions. The presence of the target in the sample is then detected bydetecting the presence of the first and second labels in at least onesame partition.

In some embodiments, the first and second labels are nucleic acid (e.g.,DNA) labels. Examples of suitable nucleic acid labels include, but arenot limited to, oligonucleotide sequences, single-stranded DNA,double-stranded DNA, RNA (e.g., mRNA or miRNA), or DNA-RNA hybrids. Insome embodiments, the nucleic acid label is about 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, or 1000 nucleotides in length. In embodimentsin which the nucleic acid label is an oligonucleotide, theoligonucleotide can be synthesized by methods known to those skilled inthe art and are commercially available. First and secondoligonucleotides used as first and second labels in embodiments hereinare generally designed such that the first oligonucleotide does nothybridize with the second oligonucleotide. As used herein, “hybridize”refers to the process of forming a double stranded nucleic acid fromjoining two complementary strands of DNA or RNA.

In certain embodiments, the nucleic acid labels are amplified. Thenucleic acid label can be amplified by, for example PCR, LCR (LigaseChain Reaction), SDA (Strand Displacement Amplification), 3SR(Self-Sustained Synthetic Reaction), TMA (Transcription-MediatedAmplification), rolling circle amplification (RCA), or hyper-branchedRCA (HRCA).

In some embodiments, the amplified nucleic acid labels are detected bydirect incorporation of a label (e.g., a fluorophore, a radioisotope, oran enzyme) into the amplified nucleic acid by using label-conjugatedprimers or nucleotides. In some embodiments, a dye that fluoresces whenit intercalates into double-stranded DNA is used to detect the amplifiednucleic acids. Exemplary intercalating dyes include, but are not limitedto, ethidium bromide, propidium iodide, EvaGreen® dye, and SYBR™ green.In some embodiments, the amplified nucleic acids are detected by using anucleic acid probe having a reporter on one end and a quencher on theother end. In some embodiments, the probe comprises a reporter-quenchercombination as employed in a TAQMAN™ probe, a molecular beacon probe, aSCORPION™ probe, a dual hybridization probe, a double-stranded probe, anECLIPSE™ probe, or a double-quenched probe (e.g., ZEN™ or TAO™Double-Quenched Probes from IDT). In some embodiments, the first andsecond nucleic acid labels are detected using different signal levels ofthe same DNA probe or intercalating dye. In embodiments using DNAprobes, for each oligo label, the DNA probe is added at a differentconcentration. In embodiments using intercalating dyes, different signalintensity for each oligo label is created by either different ampliconlength, or different concentration of primers. Droplets positive for afirst label will have a mild increase in fluorescence, droplets positivefor a second label will have an intermediate increase in fluorescence,and double positive droplets, which indicate linkage, will have thehighest fluorescence signal.

In some embodiments, the first and second labels are different enzymes,and the target is detected by detecting a product generated by each ofthe enzymes. Examples of suitable enzymes include, but are not limitedto, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase(HRP), glucose oxidase, β-galactosidase, luciferase, and an esterase.For example, a horseradish-peroxidase detection system can be used withthe fluorogenic substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP),which in the presence of hydrogen peroxide yields Resorufin, a solubleproduct that is detectable at 585 nm. An alkaline phosphatase detectionsystem can be used with the fluorogenic substrate FluoresceinDiphosphate (FDP), which yields a soluble product readily detectable at520 nm. A β-galactosidase detection system can be used with thefluorogenic substrate Resorufin β-D-Galactopyranoside (RBG), whichyields a soluble product detectable at 585 nm. An esterase detectionsystem can be used with a substrate such as fluorescein diacetate. Insome embodiments, 2, 3, 4, 5, or more affinity agents used for detectinga target molecule are each labeled with an enzyme (e.g., a firstaffinity agent labeled with a first enzyme, a second affinity agentlabeled with a second enzyme, etc.), and each affinity agent that islabeled with an enzyme is detected by detecting a distinguishableproduct generated by the enzyme.

In some embodiments, labels are linked to affinity agents bybiotin-streptavidin interaction. In some embodiments, the first andsecond labels are each linked to streptavidin and the second and thirdaffinity agents are biotinylated. The second and third affinity agentsare labeled prior to a first step of any of the methods described hereinby allowing the first and second labeled streptavidins to bind to therespective biotinylated second and third affinity agents.

The detectable label (e.g., a label as described herein) can be detectedusing any of a variety of detector devices. Exemplary detection methodsinclude optical absorbance detection (e.g., fluorescence orchemiluminescence) or radioactive detection. As a non-limiting example,a fluorescent label can be detected using a detector device equippedwith a module to generate excitation light that can be absorbed by afluorophore, as well as a module to detect light emitted by thefluorophore.

In some embodiments, the detector further comprises handlingcapabilities for the partitioned samples (e.g., droplets), withindividual partitioned samples entering the detector, undergoingdetection, and then exiting the detector. In some embodiments,partitioned samples (e.g., droplets) are detected serially while thepartitioned samples are flowing. In some embodiments, partitionedsamples (e.g., droplets) are arrayed on a surface and a detector movesrelative to the surface, detecting signal(s) at each position containinga single partition. Examples of detectors are provided in WO2010/036352, the contents of which are incorporated herein by reference.In some embodiments, detectable labels in partitioned samples aredetected serially without flowing the partitioned samples (e.g., using achamber slide).

Following acquisition of fluorescence detection data, a general purposecomputer system (referred to herein as a “host computer”) can be used tostore and process the data. Computer-executable logic can be employed toperform such functions as subtraction of background signal, assignmentof target and/or reference sequences, and qualification and/orquantification of the data. A host computer can be useful fordisplaying, storing, retrieving, or calculating diagnostic results fromthe molecular profiling; storing, retrieving, or calculating raw datafrom expression analysis; or displaying, storing, retrieving, orcalculating any sample or patient information useful in the methods ofthe present invention.

The host computer can be configured with many different hardwarecomponents and can be made in many dimensions and styles (e.g., desktopPC, laptop, tablet PC, handheld computer, server, workstation,mainframe). Standard components, such as monitors, keyboards, diskdrives, CD and/or DVD drives, and the like, can be included. Where thehost computer is attached to a network, the connections can be providedvia any suitable transport media (e.g., wired, optical, and/or wirelessmedia) and any suitable communication protocol (e.g., TCP/IP); the hostcomputer can include suitable networking hardware (e.g., modem, Ethernetcard, WiFi card). The host computer can implement any of a variety ofoperating systems, including UNIX, Linux, Microsoft Windows, MacOS, orany other operating system.

Computer code for implementing aspects of the present invention can bewritten in a variety of languages, including PERL, C, C++, Java,JavaScript, VBScript, AWK, or any other scripting or programminglanguage that can be executed on the host computer or that can becompiled to execute on the host computer. Code can also be written ordistributed in low level languages such as assembler languages ormachine languages.

The host computer system advantageously provides an interface via whichthe user controls operation of the tools. In the examples describedherein, software tools are implemented as scripts (e.g., using PERL),execution of which can be initiated by a user from a standard commandline interface of an operating system such as Linux or UNIX. Thoseskilled in the art will appreciate that commands can be adapted to theoperating system as appropriate. In other embodiments, a graphical userinterface can be provided, allowing the user to control operations usinga pointing device. Thus, the present invention is not limited to anyparticular user interface.

Scripts or programs incorporating various features of the invention asdescribed herein can be encoded on various computer readable media forstorage and/or transmission. Examples of suitable media include magneticdisk or tape, optical storage media such as compact disk (CD) or DVD(digital versatile disk), flash memory, and carrier signals adapted fortransmission via wired, optical, and/or wireless networks conforming toa variety of protocols, including the Internet.

In some embodiments, the methods further comprise quantifying the target(e.g., a protein, a protein aggregate, or a protein oligomer) bydetermining a number of partitions comprising both the first and secondlabels and determining a total number of partitions. Once a binary“yes-no” result has been determined for each of the partitions, the datafor the partitions is analyzed by an algorithm based on Poissonstatistics to quantify the amount of target in the sample. In someembodiments, the degree or amount of linkage between the two labels isproportional to the degree or amount of protein aggregation or proteinoligomerization and is used to quantify the amount of aggregation oroligomerization. An exemplary statistical method for quantifying theconcentration or amount of target or targets is described, for examplein the aforementioned WO 2010036352.

IV. Kits

In another aspect, kits for detecting a target or a plurality of targetsin a sample according to the methods described herein are provided. Insome embodiments, a kit comprises a first affinity agent linked to asolid support, a second affinity agent conjugated to a first label, anda third affinity agent conjugated to a second label, wherein each of thefirst, second, and third affinity agents specifically binds to thetarget. In embodiments in which the target is a plurality of targets,for each of the plurality of different targets in the sample, a set offirst, second, and third affinity agents is provided wherein each of thefirst, second, and third affinity agents specifically binds to thetarget. Affinity agents, affinity agents linked to a solid support, andaffinity agents conjugated to labels are described herein.

In some embodiments, the first label is a first oligonucleotide and thesecond label is a second oligonucleotide. In this embodiment, the kitcan further comprise assay components (e.g., a DNA polymerase, PCRprimers, PCR probes, dNTPs, a buffer, a PCR master mix).

In certain embodiments, the first label is a first enzyme and the secondlabel is a second enzyme. In this embodiment, the kit can furthercomprise enzyme substrates for each of the first and second enzymes.

In some embodiments, the kit comprises a chemically modified solidsupport, a first label, and a second label, wherein the first and secondlabels are each linked to streptavidin. Solid supports and labels aredescribed herein. In some embodiments, the chemically modified solidsupport can be linked to a first affinity agent supplied by the user. Insome embodiments, the first and second labeled streptavidins can bind tobiotinylated second and third affinity agents, respectively, supplied bythe user. In some embodiments, the kit comprises a streptavidin-coatedsolid support. The streptavidin-coated solid support can be linked to abiotinylated first affinity agent supplied by the user.

In some embodiments, the kit further comprises instructions for carryingout the methods described herein.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could he changed or modified toyield essentially the same or similar results.

Example 1 Quantitation of Target With a Digital Affinity Linkage AssayUsing Monoclonal Antibodies

In this example, two individual immuno-PCR assays were compared to adigital affinity linkage assay according to the invention. Purifiedhuman Decorin was used as the target protein and three differentmonoclonal Decorin antibodies were used: one antibody for the captureand the other two antibodies for detection.

Capture beads were prepared by binding biotinylated monoclonal Decorinantibody (R&D systems) to Streptavidin coated 1 μm tysolactivatedmagnetic beads (Dynabeads™ MyOne™ Streptavidin T1, Thermo FisherScientific). The beads were first washed three times with Dulbecco'sphosphate buffered saline pH 7.4 (PBS, Biological Industries, Israel).Next, the beads were mixed with biotinylated Decorin antibody at a ratioof 40 μg antibody/2×10⁹ beads in a volume of 280 μl in PBS buffer andincubated at room temperature for 2 hours. The beads were washed fromresidual antibody five times in PBS supplemented with 0.1% Bovine serumalbumin (BSA, Merck).

The detection antibodies were prepared from two different clones ofDecorin monoclonal antibodies (R&D systems) directly conjugated to aminemodified 100 base long DNA oligonucleotides (IDT) by Innova Biosciences,UK. The conjugates were purified from unbound oligonucleotides.

For each sample, 3×10⁷ capture beads were incubated with 200 μl PBSsupplemented with 0.01% tween-20 (PBS-T, Merck) and 0.1% BSA for 15minutes. The blocking solution was removed by aspiration aftermagnetizing the beads. The beads were resuspended in 25 μl of PBS-Tsupplemented with HeLa cell lysate (Ipracell, Belgium) at 25 μg/ml andspiked with concentration series of Human recombinant Decorin (R&Dsystems) at range of 100 ng/ml-10 fg/ml. The beads and samples wereincubated for 1.5 hours at room temperature on a rotator. In order tomeasure background levels, four Non-Protein Controls (NPC) were preparedin which no Decorin was spiked into the HeLa cell lysate. Following thebinding reaction, the beads were washed three times in 200 μl PBS-T.

The beads were then incubated in 50 μl of a mixture of both detectionprobes each at a concentration of 2 nM in PBS-T supplemented with 0.1%BSA and 100 ng/μl Polyadenylic acid (Merck). The probes were allowed tobind the beads for 1.5 hours at room temperature while rotating. Afterthe incubation, unbound detection probes were washed five times in PBS-Tand the beads were resuspended in 100 μl TE solution pH 8.0 (Merck). Oneμl of each sample was further diluted in 99 μl TE solution to reach beadconcentration of 3,000 beads/μl.

Finally, the amplification mix containing PCR primers, TaqMan probes,dNTPs and DNA polymerase was prepared and mixed with the samples.Droplets were generated in QX200™ Droplet Generator (Bio-Rad).

Amplification mix contained (per sample): 12 μl ddPCR supermix forprobes (Bio-Rad), 1.2 μl of 10 μM amplification primers (forward andreverse primer for each label), 0.5 μl of 10 μM FAM hydrolysis probe forthe first label, 0.5 μl of 10 μM HEX hydrolysis probe for the secondlabel (all oligonucleotides were purchased from IDT) and 6.5 μl dilutedsample.

Droplets were generated from 20 μl amplification mix, placed in ddPCRplates (Bio-Rad), sealed with Microseal ‘F’ PCR plate seal (Bio-Rad) andplaced in C1000 Thermal Cycler (Bio-Rad) for amplification. For eachsample, four PCR reactions were prepared and approximately 80,000 beadsanalyzed.

PCR cycles: hold 10 minutes at 95° C., cycle 94° C. for 30 seconds and56° C. for 1 minute 40 times, hold 98° C. for 10 minutes. Dropletfluorescence was measured by the QX200™ Droplet Reader instrument(Bio-Rad) and for each sample the number of positive and negativedroplets for each label was documented. Label concentration wascalculated by the QuantaSoft™ Software according to Poissondistribution. Assay results are calculated automatically by the softwarein the “Linkage” output by calculating the number of double positivedroplets observed above the expected number from random distribution.

Fitting the data to 4 parameter logistics (4PL) model was performedusing GraphPad Prism version 7.02, GraphPad Software, California USA.

Results of the two immuno-PCR assays and the digital affinity linkageassay are summarized in FIG. 3. The results show relatively high Limitof Detection (LOD) values for the individual labeled antibodies used inan immuno-PCR assay (triangle and square markers with dashed curve).However, when the same data is reused to calculate the linkage signal(circle markers with solid curve), the noise level is significantlyreduced, enabling detection of much lower target concentrations andextending the dynamic range by three orders of magnitude. These resultssuggest an unexpected 10 to 100 fold improvement in the signal to noiseratio when comparing results from individual immuno-PCR assays to anaffinity linkage assay.

Example 2 Quantitation of Target With a Digital Affinity Immuno-AssayLinkage Assay Using Polyclonal Antibodies

In this example, two individual immuno-PCR assays were compared to adigital affinity linkage assay for three different protein targetsaccording to the invention. Purified human antigens (PSA, EGF or IL-10)were used as the target proteins and for each target a single polyclonalantibody (targeting PSA. EGF or IL-10) was used both for the capture andfor the detection.

Capture beads were prepared for each target by binding the relevantbiotinylated poly clonal antibody (R&D systems) to Streptavidin coated 1μm tysolactivated magnetic beads (Dynabeads™ MyOne™ Streptavidin T1,Thermo Fisher Scientific). 200 μl of the beads at stock concertation of7-10×10⁹ beads/mL were transferred to an Eppendorf tube and the bufferwas removed by magnetizing the beads and aspirating the supernatant. Thebeads were resuspended in 200 μl Dulbecco's phosphate buffered saline pH7.4 (PBS, Biological Industries, Israel) supplemented with 50 nMbiotinylated polyclonal antibody (R&D systems). Next, the heads wereincubated with rotation at room temperature for 1 hour. The beads werewashed from residual antibody two times in PBS supplemented with 0.05%tween-20. Finally, the beads were resuspended in 200 μl PBS supplementedwith 0.1% Bovine serum albumin (BSA, Merck) and stored at 4° C.

Streptavidin conjugated to oligonucleotide tags was prepared in advancein the following manner. Two 5′ amino modified 70-80 bases longoligonucleotide were designed and ordered from IDT. The oligonucleotideswere designed to have no significant homology to each other or to othernatural sequences. They were separately covalently conjugated to Pierce™Streptavidin (Thermo Fisher Scientific) with the Protein-OligoConjugation Kit (TriLink BioTechnologies) according to manufacturer'sinstructions. Protein concentration and nucleotide concentration in theresulting conjugate were determined by UV absorption.

Before each experiment, the detection antibodies labeled with one of thetwo oligonucleotide tags were prepared in two separate tubes. 2 μl of 1μM biotinylated polyclonal antibodies were mixed with 2 μl of 1 μM(protein concentration) streptavidin-oligo conjugate and 36 μl PBSsupplemented with 0.1% BSA (Merck). Antibodies and conjugate wereincubated at room temperature for 1 hour and stored at 4° C.

For each sample, ˜1×10⁶ capture beads were removed from storage bufferand resuspended in 5 μl blocking solution consisting of PBS pH 7.4(Biological Industries, Israel), 1 mM D-Biotin (Thermo FisherScientific), 1 mg/mL BSA (Merck), 0.05% Tween-20 (Merck), 100 nM goatIgG (Merck), 0.1 mg/mL Salmon sperm single strand DNA (Thermo FisherScientific) and 5 nM Ethylenediaminetetraacetic acid disodium saltdihydrate (EDTA, Merck). 45 μl of blocking solution supplemented withHeLa cell lysate (Ipracell, Belgium) at 25 μg/ml, and spiked withconcentration series of human recombinant PSA/EGF/IL-10 (R&D systems) atrange of 100 ng/ml-10 fg/ml, were added to each sample of beads. Thebeads and samples were incubated for 1.5 hours at room temperature,shaking at 1200 rpm. In order to measure background levels, Non-ProteinControls (NPC) were prepared in which no antigen was spiked into theHeLa cell lysate. Following the binding reaction, the beads were washedtwice in 150 μl PBS-T (PBS with 0.05% Tween-20) by magnetizing thebeads, removing the supernatant and resuspending the beads in freshsolution.

The beads were then incubated in 50 μl of a mixture of both detectionprobes each at a concentration of 62.5 nM in blocking solution. Theprobes were allowed to bind the beads for 1.5 hours at room temperaturewhile shaking. After the incubation, unbound detection probes werewashed three times in PBS-T and the heads were resuspended in 120 μl TEsolution pH 8.0 (Merck) to a final concentration of 8,333 beads/μl.

Finally, the amplification mix containing PCR primers, TaqMan probes,dNTPs and DNA polymerase was prepared and mixed with the samples.Droplets were generated in QX200™ Droplet Generator (Bio-Rad).

Amplification mix contained (per sample): 12 μl ddPCR supermix forprobes (Bio-Rad), 1.2 μl of 10 μM amplification primers (forward andreverse primer for each label), 0.5 μl of 10 μM FAM hydrolysis probe forthe first label, 0.5 μl of 10 μM HEX hydrolysis probe for the secondlabel (all oligonucleotides were purchased from IDT), 4.2 μl doubledistilled water and 2 μl beads.

Droplets were generated from 20 μl amplification mix, placed in ddPCRplates (Bio-Rad), sealed with Microseal ‘F’ PCR plate seal (Bio-Rad) andplaced in C1000 Thermal Cycler (Bio-Rad) for amplification. For eachsample, two PCR reactions were prepared and approximately 33,000 beadsanalyzed.

PCR cycles: hold 10 minutes at 95° C., cycle 94° C. for 30 seconds and56° C. for 1 minute 40 times, hold 98° C. for 10 minutes. Dropletfluorescence was measured by the QX200™ Droplet Reader instrument(Bio-Rad) and for each sample the number of positive and negativedroplets for each label was documented. Label concentration wascalculated by the QuantaSoft™ Software according to Poissondistribution. Assay results are calculated automatically by the softwarein the “Linkage” output by calculating the number of double positivedroplets observed above the expected number from random distribution.Fitting the data to 4 parameter logistics (4PL) model was performedusing GraphPad Prism version 7.02, GraphPad Software, California USA.

Results of the three sets of immuno-assays are summarized in FIGS. 4A-4Cand Table 1.

TABLE 1 Limit of Detection values for individual Immuno-PCR assays vs.digital affinity linkage assays using the same two probes, prepared frompolyclonal antibody LOD I-PCR LOD I-PCR LOD Digital Target Antibody AAntibody B Affinity Linkage Protein probe pg/mL probe pg/mL Assay pg/mLPSA 11 9 3.5 hEGF 6.3 6.6 1.3 IL-10 200 700 <0.6

The results show improvement in the LOD values for each of the threetargets when comparing the results of the individual immuno-PCRs(triangle and square markers and dashed lines) with the linkage results(circle markers and solid lines). The background noise levels calculatedfrom the NPCs dropped 100-1000 fold between the immuno-PCR and linkageassay, resulting in improved LODs.

Example 3 Multiplex Quantitation of Several Targets With a DigitalAffinity Immuno-Assay Linkage Assay Using Polyclonal Antibodies

In this example, three immuno-assays for three different protein targetswere performed in a single reaction according to the invention. Purifiedhuman antigens (PSA, EGF and IL-10) were used as the target proteins andthree polyclonal antibodies (targeting PSA, EGF and IL-10) were usedboth for the capture and for the detection.

Capture beads were prepared separately for each target by binding therelevant biotinylated polyclonal antibody (R&D systems) to Streptavidincoated 1 μm tysolactivated magnetic beads (Dynabeads™ MyOne™Streptavidin T1, Thermo Fisher Scientific). 200 μl of the beads at stockconcertation of 7-10×10⁹ beads/mL were transferred to an Eppendorf tubeand the buffer was removed by magnetizing the beads and aspirating thesupernatant. The beads were resuspended in 200 μl Dulbecco's phosphatebuffered saline pH 7.4 (PBS, Biological Industries, Israel) supplementedwith 50 nM biotinylated polyclonal antibody (R&D systems). Next, thebeads were incubated with rotation at room temperature for 1 hour. Thebeads were washed from residual antibody two times in PBS supplementedwith 0.05% tween-20. Finally, the beads were resuspended in 66.6 μl PBSsupplemented with 0.1% Bovine serum albumin (BSA, Merck) and stored at4° C.

Streptavidin conjugated to oligonucleotide tags was prepared in advancein the following manner. For each target analyzed, two 5′ amino modified70-80 bases long oligonucleotide were designed and ordered from IDT. Theoligonucleotides were designed to have no significant homology to eachother or to other natural sequences. They were separately covalentlyconjugated to Pierce™ Streptavidin (Thermo Fisher Scientific) with theProtein-Oligo Conjugation Kit (TriLink BioTechnologies) according tomanufacturer's instructions. Protein concentration and nucleotideconcentration in the resulting conjugate were determined by Pierce™ BCAprotein assay (ThermoFisher Scientific) and UV absorption.

Before the experiment, the detection antibody for each target waslabeled with two unique oligonucleotide tags in two separate tubes. 2 μlof 1 μM biotinylated polyclonal antibodies were mixed with 2 μl of 1 μM(protein concentration) streptavidin-oligo conjugate and 36 μl PBSsupplemented with 0.1% BSA (Merck). Antibodies and conjugate wereincubated at room temperature for 1 hour and stored at 4° C.

For each sample, 1-2×10′ capture beads for each target (total of 3-6×10⁶beads) were removed from storage buffer and resuspended in 5 μl blockingsolution which was composed of PBS pH 7.4 (Biological Industries,Israel). 1 mM D-Biotin (Thermo Fisher Scientific), 1 mg/mL BSA (Merck),0.05% Tween-20 (Merck), 100 nM goat IgG (Merck), 0.1 mg/mL Salmon spermsingle strand DNA (Thermo Fisher Scientific) and 5 mMEthylenediaminetetraacetic acid disodium salt dihydrate (EDTA, Merck).45 μl of blocking solution supplemented with HeLa cell lysate (Ipracell,Belgium) at 25 μg/ml, and spiked with a mixture of human recombinantPSA, EGF and IL-10 (R&D systems) concentration series at range of 100ng/ml-10 fg/ml, was added to each sample of beads. The beads and sampleswere incubated for 1.5 hours at room temperature, shaking at 1200 rpm.In order to measure background levels, Non-Protein Controls (NPC) wereprepared in which no antigens were spiked into the HeLa cell lysate.Following the binding reaction, the beads were washed twice in 150 μlPBS-T (PBS with 0.05% Tween-20) by magnetizing the beads, removing thesupernatant and resuspending the beads in fresh solution.

The beads were then incubated in 50 μl of a mixture of six detectionprobes (two detection probes for each antigen), each probe at aconcentration of 62.5 nM in blocking solution. The probes were allowedto bind the beads for 1.5 hours at room temperature while shaking. Afterthe incubation, unbound detection probes were washed three times inPBS-T and the beads were resuspended in 120 μl TE solution pH 8.0(Merck) to a final concentration of 25,000-50,000 beads/μl.

Amplification mixes containing PCR primers, TaqMan probes, dNTPs and DNApolymerase were prepared separately for the detection of each target.Each amplification mix contained (per target per sample): 12 μl ddPCRsupermix for probes (Bio-Rad), 1.2 μl of 10 uM amplification primers(forward and reverse primer for each of the two labels), 0.5 μl of 10 μMFAM hydrolysis probe for the first label, 0.5 μl of 10 μM HEX hydrolysisprobe for the second label (all oligonucleotides were purchased fromIDT) and 4 μl double distilled water. 2 μl of the bead-boundimmuno-complexes were then added to the three different amplificationmixes and droplets were generated in QX200™ Droplet Generator (Bio-Rad).

Droplets were generated from 20 μl amplification mix, placed in ddPCRplates (Bio-Rad), sealed with Microseal ‘F’ PCR plate seal (Bio-Rad) andplaced in C1000 Thermal Cycler (Bio-Rad) for amplification. For eachsample and each target, two PCR reactions were prepared andapproximately 33,000-66,000 beads analyzed.

PCR cycles: hold 10 minutes at 95° C. cycle 94° C. for 30 seconds and56° C. for 1 minute 40 times, hold 98° C. for 10 minutes. Dropletfluorescence was measured by the QX200™ Droplet Reader instrument(Bio-Rad) and for each sample the number of positive and negativedroplets for each label was documented. Label concentration wascalculated by the QuantaSoft™ Software according to Poissondistribution. Assay results were calculated automatically by thesoftware in the “linkage” output by calculating the number of doublepositive droplets observed above the expected number from randomdistribution, for each target separately. Fitting the data to 4parameter logistics (4PL) model was performed using GraphPad Prismversion 7.02, GraphPad Software, California USA.

FIG. 5 illustrates the workflow for multiplex protein quantitation ofdifferent targets with a digital affinity linkage assay.

Results of an experiment comparing a multiplex linkage affinity assayfor three targets with three singleplex linkage affinity assays for thesame targets are summarized in FIGS. 6A-6C and Table 2.

TABLE 2 Intra-assay comparison of LOD values for three targets usingthree separate singleplex linkage assays vs. one multiplex linkageassay. LOD singleplex LOD multiplex Digital Affinity Digital AffinityTarget Linkage Assay Linkage Assay Protein pg/mL pg/mL PSA 5.1 2.5 hEGF1.7 3.1 IL-10 361 333

The results show that performing the assay in multiplex does not reducesensitivity and results in comparable LOD values for each of the threetargets when comparing to the singleplex assays.

All patents, patent applications, and other published referencematerials cited in this specification are hereby incorporated herein byreference in their entirety.

What is claimed is:
 1. A method of detecting a target in a sample, themethod comprising: contacting the sample with a first affinity agentlinked to a solid support, wherein the first affinity agent specificallybinds to the target, if present, thereby forming a target bound to thefirst affinity agent; separating the target bound to the first affinityagent from unbound material in the sample based on the presence orabsence of the solid support, thereby generating a separated samplecomprising the target bound to the first affinity agent; contacting theseparated sample with a second affinity agent comprising a first labeland a third affinity agent comprising a second label, wherein the secondand third affinity agents specifically bind to the target, therebyforming a target-labeled affinity agent complex; separating thetarget-labeled affinity agent complex from uncomplexed second and thirdaffinity agents based on the presence or absence of the solid support,thereby generating a separated target-labeled affinity agent complex;partitioning at least the separated target-labeled affinity agentcomplex into a plurality of partitions; and detecting the presence ofthe target in the sample by detecting the presence of the first andsecond labels in at least one same partition.
 2. The method of claim 1,wherein the first s a first nucleic acid abel and the second label is asecond nucleic acid label.
 3. The method of claim 2, wherein the firstand second nucleic acid labels are amplified following the partitioning.4. The method of claim 3, wherein each of the first and second nucleicacid labels are detected using an intercalating dye or a DNA probeselected from the group consisting of a TAQMAN™ probe, a SCORPION™probe, an ECLIPSE™ probe, a molecular beacon probe, a double-strandedprobe, a dual hybridization probe, and a double-quenched probe.
 5. Themethod of claim 4, wherein the first and second nucleic acid labels aredetected using different signal levels of the same DNA probe orintercalating dye.
 6. The method of claim 1, wherein the first andsecond labels are each linked to streptavidin and the second and thirdaffinity agents are biotinylated, and wherein prior to a first step ofthe method the first and second streptavidin-linked labels areconjugated to the biotinylated first and second affinity agents,respectively, by streptavidin-biotin interaction.
 7. The method of claim1, wherein the first affinity agent is biotinylated and the solidsupport is linked to streptavidin, and wherein prior to a first step ofthe method the streptavidin-linked solid support is linked to thebiotinylated first affinity agent by streptavidin-biotin interaction. 8.The method of claim 1, wherein the first label generates a first signaland the second label generates a second signal and the first signal andthe second signal are distinguishable.
 9. The method of claim 1, whereinthe solid support or the plurality of solid supports are selected fromthe group consisting of magnetic beads, non-magnetic beads, and asurface of a reaction vessel.
 10. The method of claim 1, wherein thesolid support or the plurality of solid supports comprise magnetic beadsand the target-labeled affinity agent complex is separated from theuncomplexed components in the sample using a magnet that attracts themagnetic beads in the target-labeled affinity agent complex.
 11. Themethod of claim 1, wherein the target is selected from the groupconsisting of a protein, a protein aggregate, and a protein oligomer.12. The method of claim 1, wherein the target is a complex of two ormore interacting proteins and the second and third affinity agents eachbind to one of the interacting proteins in the complex and the methodfurther comprises calculating a binding affinity (KD) of the interactingpartners.
 13. The method of claim 1, wherein each of the first, second,and third affinity agents specifically binds to a different epitope onthe target.
 14. The method of claim 1, wherein the first, second, andthird affinity agents are each selected from the group consisting of anantibody, an antibody fragment, and a nucleotide aptamer.
 15. The methodof claim 14, wherein the antibody is a monoclonal antibody and/or apolyclonal antibody.
 16. The method of claim 1, further comprisingdetermining a number of partitions comprising the first label and thesecond label, thereby quantifying the target.
 17. The method of claim 1,wherein the partitions are droplets.
 18. The method of claim 1, whereinthe sample comprises a plurality of different targets, and wherein foreach of the plurality of different targets in the sample, a set offirst, second, and third affinity agents is provided wherein each of thefirst, second, and third affinity agents specifically binds to thetarget.
 19. A kit for detecting a target in a sample, the kitcomprising: a first affinity agent linked to a solid support; a secondaffinity agent comprising a first label; and a third affinity agentcomprising a second label, wherein each of the first, second, and thirdaffinity agents specifically binds to the target.
 20. The kit of claim19, wherein the target is a plurality of targets and wherein for each ofthe plurality of different targets in the sample, a set of first,second, and third affinity agents is provided wherein each of the first,second, and third affinity agents specifically binds to the target.